Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. The hormone acts through its G-protein-coupled receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. The osteoblast produces proteases in response to PTH. One of these is matrix metalloproteinase-13 (MMP-13, collagenase-3). This enzyme has been shown to have a critical role in PTH-stimulated bone resorption and calcemic responses and endochondral bone formation. We have shown that PTH induces MMP-13 gene transcription in osteoblastic cells through a protein kinase A (PKA)-dependent pathway which regulates many of the proteins associated with the MMP-13 promoter. Our work in the last cycle of this grant has led us to conclude that, under basal conditions, the MMP-13 gene is repressed by the presence of histone deacetylase- 4 (HDAC4) bound to Runx2 at the runt domain binding site (RD at -132/-126 of the MMP-13 promoter). PTH causes the PKA-dependent phosphorylation of Runx2 and HDAC4, resulting in the release of HDAC4 from the MMP-13 promoter and its trafficking to the cytoplasm. In the nucleus, Runx2 then recruits the histone acetyl transferases (HATs), p300 and p300/CBP associated factor (PCAF). Newly transcribed and synthesized Fos/Jun subsequently bind to the activator protein-1(AP-1 at -48/-42 of the MMP-13 promoter) site and interact with p300, CBP and the proteins bound to Runx2 at the RD site;maximal transcription then ensues. Repression is re-initiated by HDAC4 re-binding to Runx2 and by the class III HDAC, SIRT1, binding to Fos/Jun at the AP-1 site. From these data of cells in culture and our preliminary data in vivo, we have developed the central hypothesis that HDACs are essential regulators of Runx2-dependent genes in the skeleton, maintaining the genes in a basally repressed state and PTH causes transient induction of transcription through dissociation of HDAC4;repression is reinitiated by re-binding of HDACs. The long-term goals of this work are to delineate the transcriptional regulatory mechanisms conveying PTH action in osteoblasts and bone. Consequently, the specific aims to test our hypothesis of this revised competing continuation proposal focus on the co-repressor proteins, HDAC4 and SIRT1, and will, 1) investigate the role of HDAC4 in regulating the expression of Runx2-dependent genes by, a. its interaction with Runx2, b. the cells expressing Runx2- dependent genes in Hdac4 null mice, c. MMP-13 and bone metabolism in Hdac4 conditional deletion mice, 2) investigate the role of SIRT1 in regulating the expression of MMP-13 by, a. its interaction with AP-1 proteins, b. MMP-13 and bone metabolism in Sirt1 conditional deletion mice. The results of this work will make major contributions to our knowledge of how PTH exerts its nuclear effects on skeletal function. Moreover, it will define how HDACs contribute to this. In so doing, the data will also provide new perspectives into treatment of disorders of calcium metabolism, cancer-associated bone disease and other bone disorders. PUBLIC HEALTH RELEVANCE: This research will investigate how a protein hormone (parathyroid hormone, PTH) is able to interact with the surface of a cell in bone and transmit signals to the cell's DNA to regulate the expression of genes involved in bone and cartilage turnover. PTH is essential for maintaining serum calcium levels, and is also being used to treat osteoporosis. The results of our research could lead to new drugs being developed, in place of PTH, to treat osteoporosis and other bone and skeletal disorders.